The large conductance Voltage- and Ca2+ dependent K+ channels (BK) are ubiquitous membrane proteins that selectively conduct K+ ions, playing a fundamental role in a multitude of physiological processes including blood flow, uresis, immunity and neurotransmission. Very recently, defects in human BK gene have been associated to forms of generalized epilepsy (Du W. et al., 2005), a disease that affects more than 40 million people worldwide. Two striking features characterize BK channels: 1) They can be activated by both membrane depolarization and intracellular Ca2+ and 2) they possess unique permeation properties, which allow a single channel conductance of 200-300 pS while maintaining a strict K+ selectivity. This proposal will focus on both these aspects of the human BK channel (hSlo). At present, the molecular events underlying Ca2+ dependent activation and the basis for the unusually large conductance in BK channel remain unknown. We propose to address these questions by combining a variety of powerful investigative tools including electrophysiology, molecular biology, and biochemistry and fluorescence spectroscopy. The three specific aims are as follows: Specific Aim 1: To investigate the Ca2+ induced conformational changes of BK channel transmembrane regions. We plan to unravel the structural changes that are taking place in BK channel voltage sensing regions during Ca2+ activation, using site directed fluorescence labeling and voltage clamp fluorometry. Specific Aim 2: To investigate the intracellular molecular events underlying BK channel Ca2+ dependent activation. Ca2+ is believed to bind at multiple locations in the intracellular C-terminus where two functional domains that regulate the K+ conductance (RCK1 and RCK2) are expected to play a major role in BK Ca2+ activation. We have designed experiments to identify and characterize the channel Ca sensors and to shed light on the chain of molecular events that, following Ca2+ binding, lead to the opening of the channel. In Specific Aim 3 we will investigate the energetics of high conductance in BK channels. We will use temperature and D2O solvent effects to investigate the distinctions between BK and lower conductance channel as a means for evaluating differences in permeation. These studies will help to understand the mechanism of operation of BK channels, particularly the molecular basis of their Ca2+ dependence and the unknown causes for the unusual large conductance. PUBLIC HEALTH RELEVANCE: The large conductance Voltage- and Ca2+-dependent K+ channels (BK) are ubiquitous cell membrane proteins that play fundamental roles in controlling blood pressure and neuronal excitability. Elevation of the intracellular Calcium concentration activates this channel. The main objective of this proposal is to investigate the molecular events that, following the binding of Calcium to intracellular structures, lead to channel opening allowing potassium flux. [unreadable] [unreadable]